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Biomedical subjects

S Harada

Publications and source records attributed to S Harada.

At least 253 records · Page 14Linked to original sources
PubMed1996

Piperastatin A, a new selective serine carboxypeptidase inhibitor produced by actinomycete. I. Taxonomy, production, isolation and biological activities.

Piperastatin A (structure, N-formyl-allo Ile-Thr-Leu-Val-Pip-Leu-Pip, Pip = hexahydropyridadine-3-carboxylic acid; molecular weight, 809), a new inhibitor of serine carboxypeptidase was discovered in the fermentation broth of Streptomyces lavendofoliae MJ908-WF13. It was purified by activated charcoal chromatography, YMC gel ODS-A chromatography and centrifugal partition chromatography (CPC) by monitoring its inhibitory activity against carboxypeptidase Y (CP-Y), and finally obtained as colourless needles. Piperastatin A is a competitive inhibitor of the enzyme with Ki = 52 +/- 6.2 nM. Piperastatin A is a highly specific inhibitor of the serine carboxypeptidases, CP-Y and platelet deamidase with little effect on related enzymes, has no antimicrobial activity and has low toxicity.

Carboxypeptidases↗
PubMed1996

Piperastatin B: a new selective serine carboxypeptidase inhibitor from Streptomyces lavendofoliae MJ908-WF13.

Piperastatin B, a new inhibitor of serine carboxypeptidase was purified from a culture broth of Streptomyces lavendofoliae MJ908-WF13 as a minor component by monitoring its inhibitory activity against carboxypeptidase Y (CP-Y). Its structure was determined to be N-formyl-Val-Thr-Leu-Val-Pip-Leu-Pip (pip: piperazic acid, hexahydropyridadine-3-carboxylic acid). Piperastatin B is a highly specific competitive inhibitor of CP-Y (Ki = 55 nM) with little effect on related enzymes and resembles the major component, piperastatin A, in these respects.

Amino Acids↗
PubMed1996

Bioactive metabolites of EM574 and EM523, erythromycin derivatives having strong gastrointestinal motor stimulating activity.

Two motilides, EM574 (N-demethyl-N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal) and EM523 (N-demethyl-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal) have strong gastrointestinal motor stimulating (GMS) activity. When administered orally to dogs, these agents showed strong GMS activity, but their plasma levels were very low and the metabolites which have been determined so far using the radio-labeled compounds show only weak GMS activity. The findings suggested that unknown bioactive metabolites might be responsible for the GMS activity. From the liver of dogs given EM574 intravenously, two bioactive metabolites, EM574 P1 and P2, were isolated by solvent extraction and chromatography as detected by contractile activity. They both showed the same UV spectra as EM574 and the molecular ion peaks at m/z 760 (MH+) and 602 (MH(+)-cladinose) in the FAB-MS. From 2D-NMR experiments, the structures of EM574 P1 and P2 were unveiled to be the 15- and 14-hydroxyl derivatives of EM574, respectively. EM523 P1 and P2 were also isolated in the same procedure. In order to prepare these bioactive metabolites, EM574 and EM523 were converted enzymatically with dog liver homogenates in the presence of co-enzymes to give the corresponding P1 and P2. The structures of the metabolites are shown in Fig 1. They exhibited stronger contractile activity in vitro and GMS activity in vivo than the parent compounds.

Animals↗
PubMed1996

Microbial conversion of EM574 and EM523, gastrointestinal motor stimulating agents.

EM574 exerts gastrointestinal motor stimulating (GMS) activity even after being converted to its metabolites P1 and P2 in dogs. These metabolites were isolated from dog liver using a series of chromatographic procedures. Their structures were determined to be the 15- and 14-hydroxyl derivatives of EM574, respectively, by spectral analysis. Large scale preparation by microbial transformation was investigated for further evaluation of the metabolites, because the amounts obtained by oxidation with dog liver homogenate were limited. Three strains of actinomycetes, Amycolatopsis tolypophorus IFO 13151, Dactylosporangium variesporum IFO 14104 and Nocardia capreola IFO 12847, were found to have the aiming oxidative potency. HPLC analysis of the crude extracts from these three cultures showed that the bioactive metabolites, EM574 P1 and P2 were produced. They were isolated from the culture broth with the other bioactive products EM574 P3 and P4. These bioactive products were prepared by large scale cultivation. EM574 P3 and P4 showed GMS activity comparable to that of EM574 P1 and P2. The structures of EM574 P3 and P4 were elucidated by spectral analysis and found to be the 3"-O-demethyl derivatives of EM574 P2 and EM574, respectively. Moreover, the absolute configuration at the C14 position of P2 was determined to be R by spectral analysis of the 6-membered cyclic carbonate of EM574 P2.

Actinomycetales↗
PubMed1996

[A case of de novo aneurysm of the distal posterior inferior cerebellar artery with intraventricular hemorrhage].

A case is presented of a de novo aneurysm of the distal posterior inferior cerebellar artery with intraventricular hemorrhage. A 67-year-old woman was admitted to our hospital with sudden onset of severe headache and loss of consciousness. Computed tomography (CT) scans showed subarachnoid hemorrhage. Angiography demonstrated three aneurysms: an aneurysm of the right vertebral-posterior inferior cerebellar artery, an aneurysm of the bifurcation of the basilar artery, and an aneurysm of the left middle cerebral artery. Considering the distribution of the hemorrhage on CT scans, we concluded that the cause of the hemorrhage was rupture of the vertebral-posterior inferior cerebellar aneurysm. The vertebral-posterior inferior cerebellar aneurysm and the middle cerebral aneurysm were successfully clipped, postoperative angiograms showing the complete clippings. At that time, however, there were no abnormal findings in the left posterior inferior cerebellar artery. Six years later, she was readmitted to our hospital because of sudden onset of headache, nausea, and vertigo. CT scans showed an intraventricular hemorrhage, especially in the fourth ventricle, although subarachnoid hemorrhage was not clearly found. Angiography revealed an aneurysm of the left distal posterior inferior cerebellar artery. She underwent clipping of the aneurysm verified by postoperative angiograms. However she had bacterial meningitis and died from pneumonia and disseminated intravascular coagulopathy. De novo aneurysms of the anterior circulation have often been reported. Carotid, ligation, smoking, the use of oral contraceptives, congenital anomalies and hypertension are major risk factors in the formation of aneurysms. A de novo aneurysm of the distal posterior inferior cerebellar artery is, however, extremely rare. In this case, the right posterior inferior cerebellar artery disappeared when the de novo aneurysm was found. So it is supposed that hemodynamic changes caused by the clipping of the right vertebral-posterior inferior cerebellar aneurysm and the left middle cerebral aneurysm had contributed to the formation of the de novo aneurysm of the left distal posterior inferior cerebellar artery. In the present study, we review the literature on the aneurysm at the distal posterior inferior cerebellar artery and on the de novo aneurysm of the vertebrobasilar artery, and discuss the radiological findings and features.

Aged↗
PubMed1996

[Efficacy of cefditoren pivoxil in the treatment of acute otitis media due to benzylpenicillin-insensitive Streptococcus pneumoniae].

Clinical and bacteriological studies were carried out on cefditoren pivoxil (CDTR-PI) granule in infantile purulent acute otitis media treated at general practice settings and the following findings were obtained: 1. Two hundred forty eight strains were isolated from 210 patients, almost all of which (81.1%) harbored the following two strains: Streptococcus pneumoniae (42.3%) and Haemophilus influenzae (38.8%). Among S. pneumoniae, benzylpenicillin (PCG)-insensitive S. pneumoniae, (PISP) or PCG-resistant S. pneumoniae (PRSP) was 36.2%, corresponding to 15.3% of all the isolates and found in 18% of all patients. 2. The bacteria in the middle ear discharge and the nasopharyngeal swabs were correlated with conformity rate of more than 80% with regard to Streptococcus pyogenes, S. pneumoniae and H. influenzae but no Staphylococcus aureus was detected simultaneously from the two sources in any of the patients. S. aureus and coagulase-negative staphylococci (CNS) were considered to be contaminants that were originated from the external auditory meatus at the time of sampling. 3. Frequencies of isolation of S. pneumoniae from different age groups were higher in a lower age group between 0 and 4 years and those of PISP or PRSP had the similar tendency. 4. Antibacterial activities were determined for CDTR and related oral antibiotics against the strains of S. pneumoniae and H. influenzae as representative isolates. CDTR had stronger antibacterial activities against both bacteria than the reference antibiotics. CDTR was found to be transferred into the otorrhea at a mean concentration of 0.58 micrograms/ml after single administration of CDTR-PI granule formulation at 3 mg(potency)/kg. 5. As for bacterial eradication efficacies in the middle ear cavity and the nasopharynx, eradication rates were higher than 80% in the middle ear cavity in all cases without large differences among bacterial species but eradication rate of PISP was 30% in the nasopharynx, and it was significantly lower than those of PSSP and other bacteria. 6. In view of clinical effectiveness, the efficacy rate was 89.4% and bacteriological effects was 92.2%; in view of safety, adverse reactions were; observed in 9.5% and the rate of usefulness was 89.4%. 7. From above-stated results, CDTR-PI was considered as a useful oral antibiotic for infantile acute otitis media including PISP infections.

Acute Disease↗
PubMed1996

Investigation of genetic risk factors associated with alcoholism.

Alcoholism is a multifactorial disease influenced by genetic-environmental interaction. Genetic variation of the receptor may be associated with alcohol dependence due to its modified function in behavioral and physiological responses. In the present study, polymorphic alleles of cholecystokinin B receptor (CCKBR), serotonin 1A receptor (HT1AR) genes, and mitochondrial DNA were analyzed. DNAs were isolated from the blood samples of 112 healthy controls and 106 alcoholics. Genetic variation was detected by SSCP analysis, followed by direct sequencing of polymerase chain reaction product as well as restriction fragment-length polymorphism. Three different mutations were found in the exon 3 sequence of CCKBR: His (CAT) at aa207-->His (CAC) (5.4%), Arg (CGC) at aa215-->His (CAC) (4.5%), and Val (GTG) at aa138-->Met (ATG) (0.9%) in controls. Genotypic distribution of alcoholics was not significantly different with that in controls. A proline (CCG) to leucine (CTG) substitution at amino acid 16 of HT1AR was found in alcoholics (4.5%) and in controls (4.7%). This mutation site of HT1AR was different in comparison with the variants reported by Nakhai et al. (Biochem Biophys. Res. Commun. 210:530-536, 1995). Analysis of the mitochondrial DNA showed that a 491 bp deletion in the sequence of ATPase exists as heteroplasmy in 58% of alcoholics, but not in controls. Heteroplasmic deletion of mitochondrial DNA may be a useful marker for alcohol abuse. Further study is undergoing to elucidate the cause and significance of this deletion in alcoholics.

Alcoholism↗
PubMed1995

Adaptation to high-salt stress in Saccharomyces cerevisiae is regulated by Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin) and cAMP-dependent protein kinase.

Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin, PP2B) of Saccharomyces cerevisiae is implicated in adaptation to high-salt conditions. Calcineurin mediates high salt-induced expression of the ENA1/PMR2 gene encoding the P-type ATPase, which is suggested to be involved in Na+ efflux. We identified the PDE1 gene encoding the low-affinity cAMP phosphodiesterase as a multicopy suppressor of the Li(+)- and Na(+)-sensitive calcineurin null mutant, suggesting that cAMP is a negative regulator of adaptation to high-salt stress. Genetic analysis indicated that calcineurin and cAMP act antagonistically in a common pathway for adaptation. The bcy1 disruption, which leads to constitutive cAMP-dependent protein kinase (PKA) activity inhibited high NaCl-induced expression of the ENA1/PMR2 gene, caused an elevation of the intracellular Na+ level and a growth defect in high-NaCl medium, all of which were analogous to the defects of a calcineurin mutant. A reduced cAMP level resulting from multiple copies of the PDE1 gene caused increased expression of the ENA1/PMR2 gene in response to high NaCl. We propose a model for the regulation of cation homeostasis, in which calcineurin antagonizes PKA to activate transcription of the ENA1/PMR2 gene in response to high-salt conditions.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Possible participation of autocrine and paracrine vascular endothelial growth factors in hypoxia-induced proliferation of endothelial cells and pericytes.

Hypoxia is the principal factor that causes angiogenesis. These experiments were conducted to explore how it induces the proliferation of vascular cells, a key step in angiogenesis. Human umbilical vein endothelial cells and bovine retinal pericytes were grown in controlled atmosphere culture chambers containing various concentrations of oxygen. The numbers of both endothelial cells and pericytes increased significantly under hypoxic conditions; the O2 concentrations that achieved maximal growth promotion were 10% for endothelial cells and 2.5% for pericytes. Quantitative reverse transcription-polymerase chain reaction analysis revealed that mRNAs coding for the secretory forms of vascular endothelial growth factor (VEGF), a mitogen specific to endothelial cells, were present in both endothelial cells and pericytes and that their levels increased significantly in the two cell types as the atmospheric O2 concentration decreased. The two genes for VEGF receptors, kinase insert domain-containing receptor (kdr) and fms-like tyrosine kinase 1 (flt1), were found to be constitutively expressed in endothelial cells, and their relative mRNA levels were ranked in that order. On the other hand, only flt1 mRNA was detected in pericytes under hypoxic conditions. Furthermore, most antisense oligodeoxyribonucleotides complementary to VEGF mRNAs efficiently inhibited DNA synthesis in endothelial cells cultured under hypoxic conditions. These results indicate that autocrine and paracrine VEGFs may take part in the hypoxia-induced proliferation of endothelial cells.

Animals↗
PubMed1995

Insulin-induced egr-1 expression in Chinese hamster ovary cells is insulin receptor and insulin receptor substrate-1 phosphorylation-independent. Evidence of an alternative signal transduction pathway.

Insulin's effects primarily are initiated by insulin binding to its plasma membrane receptor and the sequential tyrosine phosphorylation of the insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1). However, studies suggest some insulin effects, including those at the nucleus, may not be regulated by this pathway. The present study compared the levels of insulin binding, insulin receptor and IRS-1 tyrosine phosphorylation, and phosphatidylinositol 3'-kinase activity to immediate early gene c-fos and egr-1 mRNA expression in Chinese hamster ovary (CHO) cells expressing only neomycin-resistant plasmid (CHONEO), overexpressing wild type human insulin receptor (CHOHIRc) or ATP binding site-mutated insulin receptors (CHOA1018K). Insulin binding in CHONEO cells was markedly lower than that in other cell types. 10 nM insulin significantly increased tyrosine phosphorylation of insulin receptor and IRS-1 in CHOHIRc cells. Phosphorylation of insulin receptor and IRS-1 in CHONEO and CHOA1018K cells was not detected in the presence or absence of insulin. Similarly, insulin increased phosphatidylinositol 3-kinase activity only in CHOHIRc cells. As determined by Northern blot, nuclear run-on analysis, and in situ hybridization, insulin induced c-fos mRNA expression, through transcription, in CHOHIRc cells but not in CHONEO and CHOA1018K cells, consistent with previous reports. In contrast, all three cell types showed a similar insulin dose-dependent increase of egr-1 mRNA expression through transcription. These data indicated that insulin-induced egr-1 mRNA expression did not correlate with the levels of insulin binding to insulin receptor or phosphorylation of insulin receptor and IRS-1. These results suggest that different mechanisms are involved in induction of c-fos and egr-1 mRNA expression by insulin, the former by the more classic insulin receptor tyrosine kinase pathway and the latter by a yet to be determined alternative signal transduction pathway.

Adenosine Triphosphate↗
PubMed1995

Complete amino acid sequence of a zinc metalloendoprotease from Streptomyces caespitosus.

We determined the complete amino acid sequence of a zinc metalloendoprotease from Streptomyces caespitosus (ScNP). Peptide fragments obtained by digestion of Rcm-ScNP with trypsin, ScNP and endoproteinase Asp-N were purified by reverse-phase HPLC and their amino acids were analyzed using an automatic sequencer. ScNP consisted of a single polypeptide chain of 132 amino acid residues with one disulfide bond between residues 99 and 112 (M(r) 14376). Thus, the number of amino acid residues determined for this enzyme is much lower than the number of residues previously reported for metalloendoproteases. The amino acid sequence indicated that although ScNP has the zinc-binding motif. His-Glu-Xaa-Xaa-His, which is found at the active site of most zinc metalloendoproteases, it does not share overall significant similarity to the sequences of other zinc metalloendoproteases. Moreover, an analysis of the X-ray structure of ScNP at 0.2-nm resolution (Kirisu et al., unpublished results) revealed that Asp93, together with two histidine residues in the zinc-binding motif (His83 and His87) and a water molecule, is a zinc ligand. We propose that ScNP, which bears the HEXXHXXGXXD motif, represents a novel subfamily of zinc-containing metalloendoproteases.

Amino Acid Sequence↗
PubMed1995

A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin 6, transforming growth factor beta 1, or platelet-derived growth factor B gene.

Interstitial pneumonia is characterized by alveolitis with resulting fibrosis of the interstitium. To determine the relevance of humoral factors in the pathogenesis of interstitial pneumonia, we introduced expression vectors into Wistar rats via the trachea to locally overexpress humoral factors in the lungs. Human interleukin (IL) 6 and IL-6 receptor genes induced lymphocytic alveolitis without marked fibroblast proliferation. In contrast, overexpression of human transforming growth factor beta 1 or human platelet-derived growth factor B gene induced only mild or apparent cellular infiltration in the alveoli, respectively. However, both factors induced significant proliferation of fibroblasts and deposition of collagen fibrils. These histopathologic changes induced by the transforming growth factor beta 1 and platelet-derived growth factor B gene are partly akin to those changes seen in lung tissues from patients with pulmonary fibrosis and markedly contrast with the changes induced by overexpression of the IL-6 and IL-6 receptor genes that mimics lymphocytic interstitial pneumonia.

Adenoviridae↗
PubMed1995

Isolation of human T-cell leukemia virus type I from a transformed T-cell line derived spontaneously from lymphocytes of a seronegative Egyptian patient with mycosis fungoides.

Mycosis fungoides (MF) is a rare form of cutaneous T-cell lymphoma that may be associated with human T-cell leukemia virus type I (HTLV-I) infection. Using the polymerase chain reaction, the HTLV-I pX region was constantly detected in the genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) of an HTLV-I antibody-seronegative Egyptian MF patient enrolled in a study to isolate HTLV-I from North Africa. A CD4+ and interleukin-2 (IL-2) receptor-positive T-cell line was established when the phytohemagglutinin-stimulated PBMCs of that patient were maintained in IL-2-containing culture medium. The cell line (EMF) was initially IL-2 dependent and then became IL-2 independent after gradual withdrawal of the IL-2. The cells reacted positively with monoclonal antibodies specific for the HTLV-I Env or HTLV-I Gag proteins. Using the Southern blot analysis, HTLV-I provirus could be detected in the genomic DNA extracted from the EMF cells. Limited nucleotide sequence of the env region showed more than 95% homology between the EMF provirus and other known HTLV-I isolates. Western blot analysis of the cell lysates showed the expression of the HTLV-I structural proteins. These data imply that a transforming HTLV-I provirus may be present, at least in certain cases of MF, regardless of the presence or absence of the specific antibodies.

Base Sequence↗
PubMed1995

Receptor-mediated toxicity to pericytes of advanced glycosylation end products: a possible mechanism of pericyte loss in diabetic microangiopathy.

The influence of advanced glycosylation end products (AGE) on bovine retinal pericytes was investigated. When pericytes were cultured with AGE-bovine serum albumin (BSA), pericyte growth was significantly retarded in a dose-dependent manner. They also exhibited an immediate toxicity to pericytes. However, MRC-5 human fibroblasts were totally resistant to AGE-BSA. Moreover, antisense oligonucleotides complementary to mRNA coding for AGE receptor were found to reverse the AGE-induced decrease in viable pericyte number, although the mRNA level was about one order of magnitude lower in pericytes than in the fibroblasts. These results indicate that pericytes may possess a peculiar sensitivity to AGE, and that AGE ligand-receptor interactions may play an important role in the pathogenesis of pericyte loss, the principal change in diabetic microangiopathies.

Animals↗
PubMed1995

Impaired infectivity of HIV-1 after a single point mutation in the POL gene to escape the effect of a protease inhibitor in vitro.

To examine whether the mutation of protease in an HIV-1 resistant to a protease inhibitor affects the virus phenotype in vitro, the infectivity of the protease inhibitor-escape-virus was compared to that of the parent virus. In different T-cell lines, the infectivity of the escape virus was impaired by 10-fold compared to the parent virus. MT-4 cell killing by the escape virus, measured using the MTT assay, was much weaker than that by the parent virus. The escape virus contained more unprocessed Pr55gag than the parent virus. A delayed appearance of mature p24 in cells chronically infected with the escape virus was also noticed by the pulse-chase method. The same findings were obtained using pNL432 (HIV-1 DNA molecular clone) with the same mutation in the protease gene. Despite the lack of a significant difference in virus binding, less unintegrated and integrated DNA was detected in MT-4 cells infected with the escape virus compared to the parent virus. The impaired infectivity of the escape virus may be explained by the inefficient maturation of Gag proteins, due to the mutated protease, which may affect an early step in the virus life cycle.

Antigens, CD↗